The present invention relates to a cytoskeletal system, and to the use of a cytoskeletal system, composed of purified components for the identification of key proteins involved in mechanotransduction.
The present invention is based on the observation that mechanical stress induces a redistribution of cytoplasmic focal contact proteins to cytoskeletal structures.
Mechanotransduction, or force-initiated signal transduction, is the process by which cells convert mechanical stimuli into a chemical response. Mechanical forces, through the initiation of signal transduction, play a critical role in cellular development (Grill et al. 2001 Nature 409:630–633), wound healing (Timmenga et al. 1991 Br. J. Plast. Surg. 44:514–519), cell growth (Damien et al. 2000 J. Bone Miner. Res. 15:2169–2177; Chen et al. 1997 Science 276:1425–1428), tissue remodeling (Grodzinsky et al. 2000 “Cartilage tissue remodeling in response to mechanical forces,” Annu. Rev. Biomed. Eng. 2:691–713), and sensory functions, such as touch and hearing. As such, understanding the mechanism of and identifying the keyproteins in mechanotransduction may be useful for, inter alia, the treatment of wounds (e.g. treatment of burns, injuries and post-surgical lesions), the treatment of cancer through the control of cell growth, the healing of bone fractures, and the treatment of sensory disorders, such as paralysis, hearing loss and poor vision.
Although it is not well understood, the cytoskeleton, the extracellular matrix (ECM), and various traditional signaling molecules play key roles in mechanotransduction. The cytoskeleton, composed of actin microfilaments, microtubules and intermediate filaments, serves as the structural backbone for the cell and provides a mechanism for cell motility. Mechanical force induces changes in cytoskeletal structures (Komuro et al. 1989, Ryan J. Am. Acad. Der. 21:115, Dennerll et al. 1988, J Cell Biol August;107(2):665–74.), which may, in turn, be involved in the initiation of signal transduction.
Extracellular matrix (ECM) proteins, such as collagens, laminins, fibronectins, hyaluronans, and other proteoglycans, are secreted by cells and used by cells to mediate contact and adhesion to a solid substrate and also appear to be involved in cell-to-cell communication. The initiation of signal transduction by extracellular mechanical forces may be mediated through the interaction with various ECM proteins (MacKennan et al. 1998. J. Clin Invest. 101:301–310).
In addition, various components of the mitogen activated protein (MAP) kinase cascades appear to be activated by mechanical forces (Kippenburger et al. 2000 J. Invest. Dermatol. 114:408–412; Yamazaki et al. 1999 Ann. NY. Acad. Sci. 874:38–48; Ishida et al. 1996 Cir. Res. 79:310–316; Sawada et al. 2001 J. Cell Sci. 114:1221–1227.) MAP kinases are important mediators of signal transduction from the cell surface via phosphorylation cascades. Several subgroups of MAP kinases have been defined, and each manifests different substrate specificities and responds to various distinct extracellular stimuli. Cell stretching (Kippenberger et al. 2000J. Invest. Dermatol. 114:408–412; Yarnazaki et al. 1999 Ann. NY Acad. Sci. 874:38–48) or shear stress from fluid flow (Ishida et al. 1996 Cir Res. 79:310–316), both of which are types of mechanical forces, have been shown to activate the following MAP kinase pathways: extracellular signal-regulated protein kinase (ERK); c-Jun amino-terminal kinase (JNK); and p38 kinase pathways. Upstream of MAP kinases, G proteins, such as Ras and Rap1, also appear to be involved in mediating a force-initiated signal. Rap1, for example, is activated by cell stretching and inactivated by cell contraction, whereas Ras is activated by cell contraction and inactivated by cell stretching (Sawada et al. 2001 J. Cell Sci. 114:1221–1227).
Mechanical stress may also be mediated by changes in ion channel activity (Wirtz and Dobbs, 1990 Science 250:1266–1269; Pommerenke et al. 1996 Eur. J. Cell Biol. 70:157–164; Glogauer et al. 1998 J. Biol. Chem 273:1689–1698; Okuda et al. 1999 J. Biol. Chem. 274:26803–26809). However, these studies, which have shown that mechanical stress is mediated through changes in ion channel activity, do not address the role of cytoskeletal proteins which may be involved at the initial site of signal transduction at the plasma membrane.
Focal adhesion/contacts are sites found on the plasma membrane where intracellular cytoskeletal elements come into contact with ECM proteins. Proteins localized to the focal adhesions/contacts, include p125 focal adhesion kinase (FAK), paxillin, vinculin and integrins. Cells adhere tightly to the underlying substrate and the ECM proteins at focal adhesions. This adhesion is mediated by the integrin family of heterotrimeric cell surface receptors. In addition, actin filaments appear to be bundled by integrin receptors at the focal adhesions (as reviewed by Burridge et al. 1990, Cell Differ Dev Dec 2;32(3):337–42). Thus, it has been hypothesized that focal adhesions may also serve at the site of mechanotransduction. Various studies show that mechanotransduction may occur at focal adhesions through the induction of changes to integrin-cytoskeletal bonds (Choquet et al. 1997 Cell 88:39–48) and cause redistribution of proteins to focal adhesions (Balaban et al. 2001 Nat. Cell Biol. 3:466–472).
To date, no system or method is known for the quick and reliable identification of key proteins involved in mechanotransduction. Accordingly, a need exists in the art for a system or method for identifying key proteins, such as cytoplasmic proteins, involved in mechanotransduction. In one embodiment of the invention, the focal adhesion proteins, which may mediate initial transduction of mechanical stress and play a critical role in the signaling to downstream cytoplasmic molecules, are identified.